Calcium-dependent inactivation (CDI) of voltage-gated L-type Ca2+ channels is of fundamental importance in cardiac physiology. It is a major determinant of the action potential duration and thus an essential regulator of excitation-contraction coupling. Disturbed in heart failure, abnormal CDI may be a trigger for arrhythmogenesis. CDI is also a pre-eminent example of feedback inhibition, in which the very Ca2+ that permeates the channel triggers accelerated channel inactivation. This proposal aims to identify the molecular mechanisms underlying CDI by focusing on the molecular components that have recently been identified: 1) the Ca2-sensor is calmodulin (CaM), constitutively bound to the channel's pore-forming subunit alpha 1c; 2) the CDI effector in alpha 1c is the "IQ motif', a CaM binding site in the alpha 1c C-terminal tail; and 3) an EF-hand motif in the C-terminal tail serves an essential structural role.This proposal contains three specific aims: Aim 1) Determine the structural role of CaM in the regulation of inactivation Aim 2) Determine the structural role of the IQ motif in the regulation of inactivation and Aim 3) Define the role of the EF-hand(s) in CDI. The first two Aims will identify the molecular roles of CaM and the IQ motif, essential in both Ca2+-dependent and Ca2+-independent inactivation. The third Aim proposes novel hypotheses regarding the role of the controversial EF-hand motif within alpha 1c. We have identified a second EF-hand, and show preliminary results indicating that the two EF-hands form a structural pair, similar to a single lobe of CaM. Our preliminary data allow us to propose a model that incorporates how CaM, the IQ motif, and the EF-hand pair function in regulating inactivation. These novel hypotheses promise to significantly alter the current model of CDI and also provide a template for the understanding of voltage-gated channel inactivation. This work will augment our understanding of normal cardiac function and how these processes are perturbed under pathophysiological states, such as heart failure and arrhythmogenesis.